Difference between revisions of "Part:BBa K1529797:Experience"

Revision as of 16:34, 19 October 2014

Plux_CmR_RhlI

Materials and Methods

-Strain
All the samples were JM2.300 strain.

3OC12HSL-dependent CmR expression Protocol

1.Construction
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)

B. Ptet-GFP-Ptet-RhlR (psB6A1), PlacIq-CmR (pSB3K3) (Positive control)

2.Assay protocol
1.Prepare the overnight culture of cell A and B at 37°C.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture)
3. Add 30 microL of suspension in the following medium.
　　　1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)
　　　2) 3 mL of LB containing Amp and Kan + 30 microL DMSO
　　　3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)
　　　4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)

3OC12HSL-dependent C4HSL production Protocol

1.Construction
Sender
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control
Reporter
C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control
E. Ptet-RhlR(pSB6A1),　Promoter-less-GFP(pSB3K3)...Negative control

2.Assay protocol

Results

3OC12HSL-dependent CmR expression Result

We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)
Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.

• 

  Fig. 1. Plasmids

• 

  Fig. 2. Plasmids

Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.
On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL.
Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol.
As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.

3OC12HSL-dependent C4HSL production Result

Fig. 3-4-3-3 shows the fluorescence intensities generated by reporter cells.
When the reporter cell C (Plux-CmR-RhlI) was incubated in the condition (1) (the culture of the induced Customer cell), the fluorescence intensity of the reporter cell increased.
Comparing the results of condition (1) and (2), reporter cell in the supernatant of (1) had 95-fold higher fluorescence intensity.
This result indicates that Customer cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.
From this experiment, we confirmed that a new part Plux-CmR-RhlI synthesized C4HSL (RhlI) as expected.

For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki].

Applications of BBa_K1529797

User Reviews

UNIQ55f961b3d87181c4-partinfo-00000002-QINU UNIQ55f961b3d87181c4-partinfo-00000003-QINU